Medical Device

ABSTRACT

The invention is directed to an improved medical device. In particular, the invention is directed to an improved medical device having a coating comprising novel cellular factor-containing solution compositions (referred to herein as CFS compositions), such CFS compositions including conditioned medium compositions obtained from culturing extraembryonic cytokine secreting cells (ECS cells), including Amnion-derived Cellular Cytokine Solution (referred to herein as ACCS) obtained from culturing Amnion-derived Multipotent Progenitor (AMP) cells, dispersed in a polymeric coating material.

FIELD OF THE INVENTION

The field of the invention is directed to an improved medical device. Inparticular, the field of the invention is directed to an improvedmedical device having a coating comprising novel cellularfactor-containing solution compositions (referred to herein as CFScompositions), such CFS compositions including conditioned mediumcompositions obtained from culturing extraembryonic cytokine secretingcells (ECS cells), including Amnion-derived Cellular Cytokine Solution(referred to herein as ACCS) obtained from culturing Amnion-derivedMultipotent Progenitor (AMP) cells, dispersed in a polymeric coatingmaterial.

BACKGROUND OF THE INVENTION

There are approximately 25 million people in the U.S. with implanted,inserted or transcutaneous medical devices (see, for example, Hanna, K.E., Institute of Medicine (U.S.) Innovation and invention in medicaldevices: workshop summary. Washington, D.C: National Academy Press;2001. Roundtable on Research and Development of Drugs B, and MedicalDevices). The expected U.S. demand for such devices is increasing at 8%each year (see, for example, The Freedonia Group, Cleveland, Ohio, USA:Freedonia; 2010, Implantable Medical Devices to 2014—Demand and SalesForecasts, Market Share, Market Size, Market Leaders). These devicesinclude, but are not limited to, cardiac pacemakers, defibrillators,vascular grafts, dental implants, bone screws, catheters, coronaryartery stents, staples, wires, shunts and joint replacement devices.

Although many medical devices perform adequately for years,implantation, insertion or transcutaneous placement of biomaterials andmedical devices elicits a dynamic host inflammatory response (see, forexample, Anderson, J. M., et al., Foreign body reaction to biomaterials,Semin Immunol 2008; 20(2):86-100, N. Broggini, L. M. McManus, J. S.Hermann, R. Medina, R. K. Schenk, D. Buser, and D. L. Cochran,Peri-implant inflammation defined by the implant-abutment interface, JDent Res 85(5):473-478, 2006) that severely limits the integration andlong-term performance of many medical devices, including jointprostheses, chemical biosensors, electrical leads/electrodes,therapeutic delivery systems, and tissue-engineered constructs,affecting millions of patients each year.

One important example with severe life threatening consequences iscoronary stent implants. Coronary stents are frequently used subsequentto balloon angioplasty in order to maintain adequate coronary muscleblood flow. However the use of coronary stents fundamentally alters thevascular response to injury by causing an intense and prolongedinflammatory state (see, for example, Welt, F. and Rogers, C.,Arterioscler Thromb Vasc Biol, 2002; 22: 1769-1776). This inflammatorycondition causes smooth cell proliferation within the coronary arterywalls as a result of damage and removal of the endothelial cell layerthat lines the lumen of these vessels. Up to 60% of stent implantationresults in restenosis.

A medical device that is improved such that the inflammatory responseassociated with its implantation, insertion or transcutaneous placementis prevented or down-regulated would have a huge impact on the successof implanted, inserted or transcutaneous devices (A W Bridges, and A JGarcia, Anti-Inflammatory Polymeric Coatings for ImplantableBiomaterials and Devices, Journal of Diabetes Science and Technology, 2,(6):984-994, 2008). It is an object of the invention described below toprovide such an improved medical device.

BRIEF SUMMARY OF THE INVENTION

Applicants present herewith for the first time the instant inventionwhose object is to employ the anti-inflammatory properties of CFScompositions, including Amnion-derived Cytokine Solution (ACCS), inorder to prevent and/or down-regulate the inflammatory responseassociated with virtually all implanted, inserted or transcutaneouslyplaced medical devices. It is also an object of the invention todeliver, in a controlled manner, physiologically relevant inflammatoryresponse-modulating cytokines and growth factors that are capable ofpreventing and/or down-regulating the inflammatory response associatedwith virtually all implanted, inserted or transcutaneously placedmedical device. It is also an object of the invention to deliver, in acontrolled manner, physiologically relevant wound healing growth factorsand cytokines that are capable of promoting healing of tissue at themedical device implantation, insertion or transcutaneous placement sitein a patient.

Accordingly, it is an object of the instant invention to provide animproved medical device as well as methods for using the improvedmedical device, wherein the improved medical device has absorbed onto itnovel cellular factor-containing solution compositions referred toherein as CFS compositions, including ACCS. The CFS compositions,including ACCS, contain a complex and unique combination of andphysiologic levels of inflammatory response-modulating cytokines andgrowth factors found naturally in the body. The inflammatoryresponse-modulating cytokines and growth factors contained in the CFScompositions, including ACCS, are released into the local area overtime. Thus, the inflammatory response-modulating cytokines and growthfactors are delivered precisely to the area for maximal effect. Becausethe inflammatory response-modulating cytokines and growth factors arepresent in levels comparable to physiological levels found in the body,they are optimal for use in therapeutic applications which requireintervention to influence biochemical and biological processes such asthe inflammatory response. The inflammatory response-modulatingcytokines and growth factors are released slowly over time to provide acontinual, consistent physiologic level of such inflammatoryresponse-modulating cytokines and growth factors to prevent and/ordown-regulate the inflammatory response associated with implanted,inserted, or transcutaneously placed medical devices as well as tooptimize healing and/or recovery. In addition, CFS compositions,including ACCS, can be formulated prior to their absorption onto themedical device. Such formulations may includesustained-release/controlled-release/time-release/extended-releaseformulations or the addition of gelling or thickening agents to improveadsorption onto the medical device. Details on sustained-releaseformulations of CFS compositions, including ACCS, can be found in U.S.Pat. Nos. 8,058,066 and 8,088,732, both of which are incorporated hereinby reference. Further, the CFS compositions, including ACCS, may belyophilized prior to absorption onto the medical device. An importantfeature of the improved medical device described herein in that it isthe first medical device disclosed that is capable of deliveringnumerous wound healing and inflammatory response-modulating cytokinesand growth factors simultaneously, slowly and at physiologicconcentrations directly to the site of implantation, insertion ortranscutaneous placement of the medical device.

Tens of millions of medical devices are implanted, inserted ortranscutaneous placed worldwide annually into patients for medicalpurposes. Unfortunately, any foreign material implanted into the bodywill result in an inflammatory reaction which will often result inencapsulation of the implanted, inserted or transcutaneously placeddevice. These biologic responses also result in decreasing theeffectiveness of the medical devices and limiting their lifespan asuseful prostheses, stents, etc. The sustained-release of CFScompositions, including ACCS, from coated medical devices would have asignificant economic impact by increasing the effectiveness andbiocompatibility as well as function of all medical device implants,insertions or transcutaneous placements and significantly reduce thenumber of failed devices.

Accordingly, a first aspect of the invention is an improved implantablemedical device useful for surgical implantation into a subject's body,wherein the improvement to the implantable medical device comprises animplantable medical device having a coating on its surface, wherein thecoating comprises Cellular Factor-containing Solution (CFS) compositionsdispersed in a polymeric coating material.

A second aspect of the invention is an improved insertable medicaldevice useful for surgical insertion into a subject's body, wherein theimprovement to the insertable medical device comprises an insertablemedical device having a coating on its surface, wherein the coatingcomprises Cellular Factor-containing Solution (CFS) compositionsdispersed in a polymeric coating material.

A third aspect of the invention is an improved transcutaneous medicaldevice useful for insertion into a subject's body, wherein theimprovement to the transcutaneous medical device comprises antranscutaneous medical device having a coating on its surface, whereinthe coating comprises Cellular Factor-containing Solution (CFS)compositions dispersed in a polymeric coating material.

A fourth aspect of the invention is a method for down-regulating theinflammatory response that occurs following the implantation, insertionor transcutaneous placement of a medical device in a patient, the methodcomprising the step of coating the medical device with a compositioncomprising Cellular Factor-containing Solution (CFS) compositionsdispersed in a polymeric coating material.

In a specific embodiment of aspects one-four of the invention, the CFScomposition is Amnion-derived Cellular Cytokine Solution (ACCS).

In a specific embodiment of aspects one-four of the invention, the CFScomposition, including ACCS, is lyophilized prior to dispersal in thepolymeric coating material.

In another specific embodiment of aspects one-four of the invention, theACCS comprises physiological levels of VEGF, PDGF, Angiogenin, TGFβ2,TIMP-1 and TIMP-2.

In a particular embodiment of aspects one-four of the invention, thephysiological levels are about 5.0-16 ng/mL for VEGF, about 3.5-4.5ng/mL for Angiogenin, about 100-165 μg/mL for PDGF, about 2.5-2.7 ng/mLfor TGFβ2, about 0.68 μg/mL for TIMP-1 and about 1.04 μg/mL for TIMP-2.

Still another embodiment of aspects one-four of the invention is whereinthe medical device is selected from the group consisting of stents,joint replacement devices, tooth replacement devices, bone screws, bonerepair rods, bone repair plates, bone repair wires, bone repair pins,spine screws, spine rods, artificial intervertebral discs, pacemakers,shunts, contact lenses, sutures, implantable defibrillators, cochlearimplants and dental implants.

Another embodiment of aspects one-four of the invention is wherein thesubject is a human subject or a non-human animal subject.

FIGURE LEGENDS

FIG. 1—Sustained-release of ACCS from Zimaloy® prosthetic hipreplacement device and from a section of a Dacron® vascular graft.

DEFINITIONS

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning. Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

As used herein, the term “extraembryonic cytokine-secreting cells” or“ECS cells” means a population of cells derived from the extraembryonictissue which have the characteristic of secreting VEGF, Angiogenin, PDGFand TGFβ2 and the MMP inhibitors TIMP-1 and/or TIMP-2 at physiologicallyrelevant levels in a physiologically relevant temporal manner into theextracellular space or into the surrounding culture media. ECS cellshave not been cultured in the presence of any non-human animalmaterials, making them and cell products derived from them suitable forhuman clinical use as they are not xeno-contaminated. ECS cells may beselected from populations of cells and compositions described in thisapplication and in US2003/0235563, US2004/0161419, US2005/0124003, U.S.Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067,60/813,759, U.S. application Ser. No. 11/333,849, U.S. application Ser.No. 11/392,892, PCTUS06/011392, US2006/0078993, PCT/US00/40052, U.S.Pat. No. 7,045,148, US2004/0048372, and US2003/0032179, the contents ofwhich are incorporated herein by reference in their entirety. ECS cellshave previously been referred to as TSE cells.

As used herein, the term “Amnion-derived Multipotent Progenitor cell” or“AMP cell” means a specific population of cells that are epithelialcells derived from the amnion. AMP cells have the followingcharacteristics. They have not been cultured in the presence of anynon-human animal materials, making them and cell products derived fromthem suitable for human clinical use as they are not xeno-contaminated.AMP cells are cultured in basal medium supplemented with human serumalbumin. In a preferred embodiment, the AMP cells secrete the cytokinesVEGF, Angiogenin, PDGF and TGFβ2 and the MMP inhibitors TIMP-1 and/orTIMP-2. The physiological range of the cytokine or cytokines in theunique combination is as follows: about 5-16 ng/mL for VEGF, about3.5-4.5 ng/mL for Angiogenin, about 100-165 μg/mL for PDGF, about2.5-2.7 ng/mL for TGFβ2, about 0.68 μg/mL for TIMP-1 and about 1.04μg/mL for TIMP-2. The AMP cells may optionally express Thymosin β4. AMPcells grow without feeder layers, do not express the protein telomeraseand are non-tumorigenic. AMP cells do not express the hematopoietic stemcell marker CD34 protein. The absence of CD34 positive cells in thispopulation indicates the isolates are not contaminated withhematopoietic stem cells such as umbilical cord blood or embryonicfibroblasts. Virtually 100% of the cells react with antibodies to lowmolecular weight cytokeratins, confirming their epithelial nature.Freshly isolated amnion-derived cells, from which AMP cells areisolated, will not react with antibodies to the stem/progenitor cellmarkers c-kit (CD117) and Thy-1 (CD90). Several procedures used toobtain cells from full term or pre-term placenta are known in the art(see, for example, US 2004/0110287; Anker et al., 2005, Stem Cells22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn. 172:493-500).However, the methods used herein provide improved compositions andpopulations of cells.

By the term “animal-free” when referring to certain compositions, growthconditions, culture media, etc. described herein, is meant that nonon-human animal-derived materials, such as bovine serum, proteins,lipids, carbohydrates, nucleic acids, vitamins, etc., are used in thepreparation, growth, culturing, expansion, storage or formulation of thecertain composition or process. By “no non-human animal-derivedmaterials” is meant that the materials have never been in or in contactwith a non-human animal body or substance so they are notxeno-contaminated. Only clinical grade materials, such as recombinantlyproduced human proteins, are used in the preparation, growth, culturing,expansion, storage and/or formulation of such compositions and/orprocesses.

By the term “expanded”, in reference to cell compositions, means thatthe cell population constitutes a significantly higher concentration ofcells than is obtained using previous methods. For example, the level ofcells per gram of amniotic tissue in expanded compositions of AMP cellsis at least 50 and up to 150 fold higher than the number of amnionepithelial cells in the primary culture after 5 passages, as compared toabout a 20 fold increase in such cells using previous methods. Inanother example, the level of cells per gram of amniotic tissue inexpanded compositions of AMP cells is at least 30 and up to 100 foldhigher than the number of amnion epithelial cells in the primary cultureafter 3 passages. Accordingly, an “expanded” population has at least a 2fold, and up to a 10 fold, improvement in cell numbers per gram ofamniotic tissue over previous methods. The term “expanded” is meant tocover only those situations in which a person has intervened to elevatethe number of the cells.

As used herein, the term “passage” means a cell culture technique inwhich cells growing in culture that have attained confluence or areclose to confluence in a tissue culture vessel are removed from thevessel, diluted with fresh culture media (i.e. diluted 1:5) and placedinto a new tissue culture vessel to allow for their continued growth andviability. For example, cells isolated from the amnion are referred toas primary cells. Such cells are expanded in culture by being grown inthe growth medium described herein. When such primary cells aresubcultured, each round of subculturing is referred to as a passage. Asused herein, “primary culture” means the freshly isolated cellpopulation.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium. Examples of methods of preparingconditioned media are described in U.S. Pat. No. 6,372,494 which isincorporated by reference in its entirety herein.

As used herein, the term “Amnion-derived Cellular Cytokine Solution” or“ACCS” means conditioned medium that has been derived from AMP cellsthat have been cultured in basal media supplemented with human serumalbumin and recombinant human EGF.

The term “physiological level” as used herein means the level that asubstance in a living system is found and that is relevant to the properfunctioning of a biochemical and/or biological process.

As used herein, the term “solution” as used in “Amnion-derived CellularCytokine Solution” means a liquid containing dispersed components, i.e.cytokines. The dispersed components may be fully solubilized, partiallysolubilized, suspended or otherwise dispersed in the liquid. Suitableliquids include, but are not limited to, water, osmotic solutions suchas salt and/or sugar solutions, cell culture media, and other aqueous ornon-aqueous solutions.

The term “lysate” as used herein refers to the composition obtained whencells, for example, AMP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by sonication, by use ofdetergents, such as EDTA, or by enzymatic digestion using, for example,hyaluronidase, dispase, proteases, and nucleases. In some instances, itmay be desirable to lyse the cells and retain the cellular membraneportion and discard the remaining portion of the lysed cells.

As used herein, the term “pooled” means a plurality of compositions thathave been combined to create a new composition having more constant orconsistent characteristics as compared to the non-pooled compositions.For example, pooled ACCS have more constant or consistentcharacteristics compared to non-pooled ACCS. Examples of pooledcompositions include “SP pools” (more than one ACCS collection/oneplacenta), “MP1 pools” (one ACCS collection/placenta, multipleplacentas), and “MP2 pools” (more than one ACCS collection/placenta,multiple placentas).

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

The term “cell product” or “cell products” as used herein refers to anyand all substances made by and secreted from a cell, including but notlimited to, protein factors (i.e. growth factors, differentiationfactors, engraftment factors, cytokines, morphogens, proteases (i.e. topromote endogenous cell delamination, protease inhibitors),extracellular matrix components (i.e. fibronectin, etc.).

As used herein, the term “inflammatory response-modulating cytokines andgrowth factors” means physiologically relevant cytokines and growthfactors that are capable of preventing and/or down-regulating theinflammatory response associated with virtually all medical deviceimplants.

The term “therapeutically effective amount” means that amount of atherapeutic agent necessary to achieve a desired physiological effect(i.e. prevent and/or down-regulate an inflammatory response associatedwith an implanted/inserted medical device).

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents.

As used herein, the term “agent” means an active agent or an inactiveagent. By the term “active agent” is meant an agent that is capable ofhaving a physiological effect when administered to a subject.Non-limiting examples of active agents include growth factors,cytokines, antibiotics, cells, conditioned media from cells, etc. By theterm “inactive agent” is meant an agent that does not have aphysiological effect when administered. Such agents may alternatively becalled “pharmaceutically acceptable excipients”. Non-limiting examplesinclude time-release capsules and the like.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, epidural,intracerebral and intrasternal injection or infusion.

The term “enteral administration” and “administered enterally” areart-recognized and refer to modes of administration other than enteraland topical administration, usually by oral or rectal routes.

The term “topical administration” and “administered topically” areart-recognized and refer to modes of administration other thanparenteral and enteral administration, usually by application to theskin.

The term “adsorb” as used herein refers to the act of a liquid, gas, ora dissolved substance accumulating on the surface of a solid.

The terms “sustained-release”, “extended-release”, “time-release”,“controlled-release”, or “continuous-release” as used herein means anagent, typically a therapeutic agent or drug, that is released overtime.

The terms “bioerodable” or “bioerosion” as used herein mean acombination of physical (i.e. dissolution) and chemical (i.e. chemicalbond cleavage) processes that result in the breakdown of a sub stance.

The term “biodegradable” or “biodegradation” as used herein means abiological agent (i.e. an enzyme, microbe or cell) is responsible forthe breakdown of a substance.

The terms “bioresporbable” or “bioabsorptable” as used herein mean theremoval of a breakdown product by cellular activity (i.e. phagocytosis).The term “nonabsorbable” as used herein means that a substance is notbroken down by a chemical process.

As used herein, the term “medical device” means an instrument,apparatus, implant, in vitro reagent, or similar or related article thatis used to diagnose, prevent, or treat disease or other conditions, anddoes not achieve its purposes through chemical action within or on thebody (which would make it a drug). Medical device includes implantedmedical device, implantable medical device, inserted medical device,insertable medical device, and medical devices placed on a body surface(i.e., on skin, cornea, etc.).

As used herein, the term “transcutaneous medical device” means a medicaldevice that is implanted in the body and exits externally through theskin.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

As used herein, a “wound” is any disruption, from whatever cause, ofnormal anatomy (internal and/or external anatomy) including but notlimited to traumatic injuries such as mechanical (i.e. contusion,penetrating), thermal, chemical, electrical, concussive and incisionalinjuries; elective injuries such as operative surgery and resultantincisional hernias, fistulas, etc.; acute wounds, chronic wounds,infected wounds, and sterile wounds, as well as wounds associated withdisease states (i.e. ulcers caused by diabetic neuropathy or ulcers ofthe gastrointestinal or genitourinary tract). A wound is dynamic and theprocess of healing is a continuum requiring a series of integrated andinterrelated cellular processes that begin at the time of wounding andproceed beyond initial wound closure through arrival at a stable scar.These cellular processes are mediated or modulated by humoral substancesincluding but not limited to cytokines, lymphokines, growth factors, andhormones. In accordance with the subject invention, “wound healing”refers to improving, by some form of intervention, the natural cellularprocesses and humoral substances of tissue repair such that healing isfaster, and/or the resulting healed area has less scaring and/or thewounded area possesses tissue strength that is closer to that ofuninjured tissue and/or the wounded tissue attains some degree offunctional recovery.

As used herein the term “standard animal model” refers to anyart-accepted animal model in which the compositions of the inventionexhibit efficacy.

DETAILED DESCRIPTION

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

The anti-inflammatory properties of Amnion-derived Cytokine Solution(ACCS) are currently being used to assist in wound healing in humanclinical trials. ACCS contains more than 200 proteins, cytokines, andgrowth factors in solution and has been shown to reduce inflammation inseveral animal models (see Example below). In addition, ACCS ispresently being tested in several human clinical trials involvinginflammation resulting from radiation burns and skin grafts in diabeticpatients being treated for burns.

As described above, implantation of any medical device results localinflammation at the implant site. In the case of coronary stents,leukocyte recruitment and infiltration occur at sites of vascular injurywhere the endothelial cells lining the vessels have been denuded andplatelets and fibrin have been deposited. In vivo studies have shownthat leukocytes and platelets co-localize at sites of hemorrhage, withinatherosclerotic and post-angioplasty restenotic lesions, and in areas ofischemia/reperfusion injury. This interaction between platelets andleukocytes appears to be critical to the inflammatory response. Monocytechemo-attractant protein (MCP)-1 participates in the recruitment ofmonocytes as well as basophils and certain activated T cells.Interleukin (IL)-8, plays a critical role in the recruitment ofleukocytes to areas of vascular injury. It is well known that IL-8 is apivotal cytokine in the recruitment of neutrophils. The nonspecificinflammatory marker, C-reactive protein, has been shown to beup-regulated after stent placement.

This invention is directed to the incorporation of CFS compositions,including ACCS, into or onto any or allimplantable/insertable/transcutaneous medical devices in order to effectthe sustained delivery of CFS compositions, including ACCS, and theiranti-inflammatory properties at the implantation/insertion site. Thisnovel improvement to the medical devices will impart greaterbiocompatibility and safety to these devices.

The CFS composition, including ACCS, maybe coated directly onto themedical devices or suspended in a polymer solution formed by dissolvinga pharmaceutically acceptable polymer (e.g. ethylene vinyl acetatecopolymer, EVAc) in a volatile organic solvent such as dichloromethane.The medical device is then submerged into the polymer coating andremoved. The organic solvent is allowed to evaporate, leaving a polymercoating containing the polymer with the CFS compositions' proteins,including ACCS proteins, dispersed in dry powdered form within thecoating. Such a CFS composition/polymer coating would be capable ofsustained-delivery of the CFS composition from the medical devices.Furthermore, as the CFS composition incorporated into or onto themedical devices will be in lyophilized or freeze-dried form, the releaseof the CFS composition will only occur upon contact with an aqueoussolution capable of solubilizing and releasing the CFS composition.

It is also possible to coat any such medical device by spraying the CFScomposition/polymer solution onto the medical device and allowing theorganic solvent to evaporate.

Therefore, local absorption of a CFS composition, including ACCS, fromthe coated medical device would be expected to effectively deliver the200 proteins, cytokines, and growth factors, includinginflammatory-response modulating growth factors and cytokines, to thesites of inflammation associated with implantation/insertion of amedical device in a sustained fashion. It should be understood that therelease kinetics of the CFS compositions from the coating can be alteredby adjusting the drug loading, drug particle size, device geometry, etc.in order to achieve first order, zero order or other release kineticprofiles.

Obtaining and Culturing of Cells

ECS Cells

Various methods for isolating cells from extraembryonic tissue, whichmay then be used to produce the ECS cells of the instant invention aredescribed in the art (see, for example, US2003/0235563, US2004/0161419,US2005/0124003, U.S. Provisional Application Nos. 60/666,949,60/699,257, 60/742,067, 60/813,759, U.S. application Ser. No.11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179).

Identifying ECS Cells

Once extraembryonic tissue is isolated, it is necessary to identifywhich cells in the tissue have the characteristics associated with ECScells (see definition above). For example, cells are assayed for theirability to secrete VEGF, Angiogenin, PDGF and TGFβ2 and the MMPinhibitors TIMP-1 and/or TIMP-2 into the extracellular space or intosurrounding culture media. In some instances, it may be difficult orimpossible to detect certain factors using standard assays. This may bebecause certain factors are secreted by the cells at physiologicallevels that are below the level of detection by the assay methods. Itmay also be that the factor(s) is being utilized by the ECS cell and/orby other local cells, thus preventing accumulation at detectable levelsusing standard assays. It is also possible that the temporal manner inwhich the factors are secreted may not coincide with the timing ofsampling.

AMP cell compositions are prepared using the steps of a) recovery of theamnion from the placenta, b) dissociation of the epithelial cells fromthe amniotic membrane using a protease, c) culturing of the cells in abasal medium with the addition of a naturally derived or recombinantlyproduced human protein (i.e. human serum albumin) and no non-humananimal protein; d) selecting AMP cells from the epithelial cell culture,and optionally e) further proliferation of the cells, optionally usingadditional additives and/or growth factors (i.e. recombinant human EGF).Details are contained in U.S. Pat. No. 8,278,095, issued Oct. 2, 2012,U.S. Pat. No. 8,058,066, issued Nov. 15, 2011 and U.S. Pat. No.8,088,732, issued Jan. 3, 2012, all of which are incorporated herein byreference.

Culturing of the AMP Cells

The cells are cultured in a basal medium. Such medium includes, but isnot limited to, EPILIFE® culture medium for epithelial cells (CascadeBiologicals), OPTI-PRO™ serum-free culture medium, VP-SFM serum-freemedium, IMDM highly enriched basal medium, KNOCKOUT™ DMEM low osmolalitymedium, 293 SFM II defined serum-free medium (all made by Gibco;Invitrogen), HPGM hematopoietic progenitor growth medium, Pro 293S-CDMserum-free medium, Pro 293A-CDM serum-free medium, UltraMDCK™ serum-freemedium (all made by Cambrex), STEMLINE® T-cell expansion medium andSTEMLINE® II hematopoietic stem cell expansion medium (both made bySigma-Aldrich), DMEM culture medium, DMEM/F-12 nutrient mixture growthmedium (both made by Gibco), Ham's F-12 nutrient mixture growth medium,M199 basal culture medium (both made by Sigma-Aldrich), and othercomparable basal media. Such media should either contain human proteinor be supplemented with human protein. As used herein a “human protein”is one that is produced naturally or one that is produced usingrecombinant technology. “Human protein” also is meant to include a humanderivative or preparation thereof, such as human serum, which containshuman protein. In specific embodiments, the basal media is IMDM highlyenriched basal medium, STEMLINE® T-cell expansion medium or STEMLINE® IIhematopoietic stem cell expansion medium, or OPTI-PRO™ serum-freeculture medium, or combinations thereof and the human protein is humanserum albumin is at least 0.5% and up to 10%. In particular embodiments,the human serum albumin is from about 0.5 to about 2%. In a specificembodiment the human albumin is at 0.5%. The human albumin may come froma liquid or a dried (powder) form and includes, but is not limited to,recombinant human serum albumin, PLASBUMIN® normal human serum albuminand PLASMANATE® human blood fraction (both made by TalecrisBiotherapeutics).

The invention further contemplates the use of any of the above basalmedia wherein animal-derived proteins are replaced with recombinanthuman proteins and animal-derived serum, such as BSA, is replaced withhuman serum albumin. In preferred embodiments, the media is serum-freein addition to being animal-free.

Optionally, other factors are used. In one embodiment, epidermal growthfactor (EGF) at a concentration of between 0-1 μg/mL is used. In apreferred embodiment, the EGF concentration is around 10-20 ng/mL. Allsupplements are clinical grade.

Generation of CFS Compositions, Including ACCS

ECS conditioned medium—is obtained as described below for ACCS, exceptthat ECS cells are used.

Generation of ACCS

The AMP cells of the invention can be used to generate ACCS. In oneembodiment, the AMP cells are isolated as described herein and 1×10⁶cells/mL are seeded into T75 flasks containing between 5-30 mL culturemedium, preferably between 10-25 mL culture medium, and most preferablyabout 10 mL culture medium. The cells are cultured until confluent, themedium is changed and in one embodiment the ACCS is collected 1 daypost-confluence. In another embodiment the medium is changed and ACCS iscollected 2 days post-confluence. In another embodiment the medium ischanged and ACCS is collected 4 days post-confluence. In anotherembodiment the medium is changed and ACCS is collected 5 dayspost-confluence. In a preferred embodiment the medium is changed andACCS is collected 3 days post-confluence. In another preferredembodiment the medium is changed and ACCS is collected 3, 4, 5, 6 ormore days post-confluence. Skilled artisans will recognize that otherembodiments for collecting ACCS from AMP cell cultures, such as usingother tissue culture vessels, including but not limited to cellfactories, flasks, hollow fibers, or suspension culture apparatus, orcollecting ACCS from sub-confluent and/or actively proliferatingcultures, are also contemplated by the methods of the invention. It isalso contemplated by the instant invention that the ACCS becryopreserved following collection. It is also contemplated by theinvention that ACCS be lyophilized following collection. It is alsocontemplated by the invention that ACCS be formulated forsustained-release following collection. Skilled artisans are familiarwith cryopreservation lyophilization, and sustained-release formulationmethodologies.

The ACCS of the invention is characterized by assaying forphysiologically relevant cytokines secreted in the physiologicallyrelevant range of about 5-16 ng/mL for VEGF, about 3.5-4.5 ng/mL forAngiogenin, about 100-165 μg/mL for PDGF, about 2.5-2.7 ng/mL for TGFβ2,about 0.68 μg/mL for TIMP-1 and about 1.04 μg/mL for TIMP-2.

It is also contemplated by the invention that ACCS, including pooledACCS, be concentrated prior to use. The appropriate level ofconcentration required will be dependent upon the intended use andtherefore will need to be empirically determined.

The compositions of the invention can be prepared in a variety of waysdepending on the intended use of the compositions. For example, acomposition useful in practicing the invention may be a liquidcomprising an agent of the invention, i.e. CFS compositions, includingACCS, in solution, in suspension, or both (solution/suspension). Theterm “solution/suspension” refers to a liquid composition where a firstportion of the active agent is present in solution and a second portionof the active agent is present in particulate form, in suspension in aliquid matrix. A liquid composition also includes a gel. The liquidcomposition may be aqueous or in the form of an ointment, salve, cream,or the like.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Protein based polymers such as gelatin arealso contemplated as useful matrix forming materials. An aqueoussuspension or solution/suspension of the present invention is preferablyviscous or muco-adhesive, or even more preferably, both viscous andmuco-adhesive. Hydrophobic crystalline or amorphous polymers such asethylene vinyl acetate copolymer (EVAc) maybe useful as a matrix formingmaterials. Polymers such as polylactic acid (PLA), polycaprolactone,polylactide/glycolide (PLGA), polyothroesters, polyanhydrides etc. areexamples of biodegradable polymers that may be useful matrix formingmaterials.

Alternative Formulation of CFS Compositions, Including ACCS

The CFS compositions, including ACCS, may be formulated assustained-release/controlled-release/timed-release/extended-releasecompositions. Skilled artisans are familiar with methodologies to createsuch compositions of therapeutic agents, including protein-basedtherapeutic agents such as CFS compositions, including ACCS.Sustained-release/controlled-release/timed-release CFS compositions,including ACCS, may be made by any of the methods described herein, aswell. In addition, other sustained-release methodologies familiar toskilled artisans, while not specifically described herein, are alsosuitable for use with the CFS compositions, including ACCS.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions of CFScompositions, including ACCS, and a pharmaceutically acceptable carrier.The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or other country'sregulatory agency, or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the composition is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil, biocompatible polymers such asethylene vinyl acetate copolymer, polylactic acid,polylactide/glycolide, polyhydroxymethyl methacrylate, carboxymethylcellulose, polyethylene glycol, poloxamer, and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin, and still others are familiar to skilledartisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

One of skill in the art may readily determine the appropriateconcentration, or dose, of the CFS compositions, including ACCS, for aparticular purpose. The skilled artisan will recognize that a preferreddose is one which produces a therapeutic effect, such as preventingand/or down-regulating the inflammatory response associated with theimplantation, insertion or transcutaneous placement of a medical device,in a patient. Of course, proper doses of the CFS compositions, includingACCS, will require empirical determination at time of use based onseveral variables including but not limited to the type of medicaldevice being used; patient age, weight, sex, health; other medicationsand treatments being administered to the patient; and the like.

In further embodiments of the present invention, it may be desirable toco-administer other agents, including active agents and/or inactiveagents, with the coated medical device. Active agents include but arenot limited to cytokines, chemokines, antibodies, inhibitors,antibiotics, anti-fungals, anti-virals, immunosuppressive agents, andthe like. Inactive agents include carriers, diluents, stabilizers,gelling agents, thickening agents (i.e. human serum albumin, hyaluronicacid), delivery vehicles, ECMs (natural and synthetic), scaffolds,collagen, and the like. When the medical device is administeredconjointly with other pharmaceutically active agents, even less of theCFS compositions, including ACCS, on the medical device may be needed tobe therapeutically effective.

Skilled artisans will recognize that any and all of the standard methodsand modalities for implanting, inserting or transcutaneous placement ofmedical devices currently in clinical practice and clinical developmentare suitable for practicing the methods of the invention. Routes ofadministration, formulation, co-administration with other agents (ifappropriate) and the like are discussed in detail elsewhere herein.

Exemplary Therapeutic Uses of a Coated Medical Device

Artificial Eye Lenses (Pseudophakos), number of procedures: 2.582million, total annual expenditure: $8 billion-$10 billion, average costper eye: $3,200-$4,500, depending on lens type. Major manufacturersinclude Alcon Laboratories/Novartis, Abbott Laboratories, and Bausch &Lomb.

Ear Tubes (Tympanostomy Tubes), number of procedures: 715,000, totalannual expenditure: $1 billion-$2 billion, average cost per procedure:$1,000-$4,500.

Coronary Stents, number of procedures: 560,000, total annualexpenditure: $7.5 billion, average cost per procedure: $13,000. Majormanufacturers include Boston Scientific and Abbott Laboratories.

Artificial Knees, number of procedures: 543,000, total annualexpenditure: $12 billion, average cost per procedure: $22,000. Majormanufacturers include Zimmer, Depuy/J&J, Stryker, and Biomet, Smith &Nephew

Metal Screws, Pins, Plates, and Rods (Traumatic Fracture Repair), numberof procedures: 453,000, total annual expenditure: $4.5 billion, averagecost per procedure: $2,000-$20,000. Major manufacturer includes Synthes.

IUDs (Intra-Uterine Devices), number of procedures: 425,000, totalannual expenditure: $340 million, average cost per procedure: $800.Major manufacturers include Teva Pharmaceutical Industries and BayerHealthCare.

Implantable Dental Devices, number of procedures: 2 million dentalimplants, total annual expenditure: $6 billion, average cost perprocedure: $2,500, Major players: Nobel Biocare and Straumann.

Spine Screws, Rods, and Artificial Discs (Spinal Fusion Hardware),number of procedures: 413,000, total annual expenditure: $10 billion,average cost per procedure: $25,000. Major manufacturer includesMedtronic.

Breast Implants, number of procedures: 366,000, total annualexpenditure: $992 million, average cost per procedure: $3,351. Majormanufacturers include Allergan and Mentor.

Heart Pacemakers, number of procedures: 235,567, total annualexpenditure: $4.5 billion, average cost per procedure: $20,000. Majormanufacturers include Medtronic, St. Jude Medical, and BostonScientific.

Artificial Hips, number of procedures: 230,000, total annualexpenditure: $10.5 billion, average cost per procedure: $45,000. Majormanufacturers include Zimmer, Stryker, DePuy/J&J, Biomet, and WrightMedical.

Implantable Cardioverter Defibrillators, number of procedures: 133,262,total annual expenditure: $5.5 billion, average cost per procedure:$40,000. Major manufacturers include Medtronic, St. Jude Medical, andBoston Scientific.

Contact lenses, staples and other devices not explicitly mentioned arealso contemplated by the invention described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions and methods of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of AMP Cell Compositions

Amnion epithelial cells were dissociated from starting amniotic membraneusing the dissociation agents PXXIII The average weight range of anamnion was 18-27 g. The number of cells recovered per g of amnion wasabout 10-15×10⁶ for dissociation with PXXIII

Method of Obtaining Selected AMP Cells

Amnion epithelial cells were plated immediately upon isolation from theamnion. After ˜2 days in culture non-adherent cells were removed and theadherent cells were kept. This attachment to a plastic tissue culturevessel is the selection method used to obtain the desired population ofAMP cells. Adherent and non-adherent AMP cells appear to have a similarcell surface marker expression profile but the adherent cells havegreater viability and are the desired population of cells. Adherent AMPcells were cultured in basal medium supplemented with human serumalbumin until they reached 120,000-150,000 cells/cm². At this point, thecultures were confluent. Suitable cell cultures will reach this numberof cells between ˜5-14 days. Attaining this criterion is an indicator ofthe proliferative potential of the AMP cells and cells that do notachieve this criterion are not selected for further analysis and use.Once the AMP cells reached ˜120,000-150,000 cells/cm², they werecollected and cryopreserved. This collection time point is called p0.

Example 2 Generation of ACCS

The AMP cells of the invention can be used to generate ACCS, includingpooled ACCS. The AMP cells were isolated as described above and about1×10⁶ cells/mL were seeded into T75 flasks containing ˜10 mL culturemedium as described above. The cells were cultured until confluent, themedium was changed and ACCS was collected 3 days post-confluence.Optionally, the ACCS is collected again after 3 days, and optionallyagain after 3 days. Skilled artisans will recognize that otherembodiments for collecting ACCS from confluent cultures, such as usingother tissue culture vessels, including but not limited to cellfactories, flasks, hollow fibers, or suspension culture apparatus, etc.are also contemplated by the methods of the invention (see DetailedDescription above). It is also contemplated by the instant inventionthat the ACCS be cryopreserved, lyophilized, irradiated or formulatedfor sustained-release following collection. It is also contemplated thatACCS be collected at different time points (see Detailed Description fordetails).

Example 3 Generation of Pooled ACCS

ACCS was obtained essentially as described above. In certainembodiments, ACCS was collected multiple times from an AMP culturederived from one placenta and these multiple ACCS collections werepooled together. Such pools are referred to as “SP pools” (more than oneACCS collection/one placenta). In another embodiment, AMP cultures werederived from several placentas, i.e. from 5 or 10 placentas. The AMPcells from each placenta were cultured and one ACCS collection from eachculture was collected and then they were all pooled. These pools aretermed “MP1 pools” (one ACCS collection/placenta, multiple placentas).In yet another embodiment, AMP cell cultures were derived from severalplacentas, i.e. from 5 or 10 placentas. The AMP cells from each placentawere cultured and more than one ACCS collection was performed from eachAMP cell culture and then pooled. These pools are termed “MP2 pools”(more than one ACCS collection/placenta, multiple placentas).

Example 4 Release of ACCS from an Implantable Medical Device

Preparation of ACCS polymer suspension: Ethylene vinyl acetate copolymer(ELVAX 40W, DuPont, Wilmington, Del.) was dissolved in dichloromethaneto form a 10% (wt/vol) solution. Twenty-five mg of lyophilized ACCS wasweighed and suspended in 2 mL of the polymer solution.

Experiment 1

A portion of a Zimaloy® prosthetic replacement hip device was dippedinto the ACCS-ethylene vinyl acetate copolymer suspension to partiallycoat the ball joint insertion end and the pointed end of the device. Thedevice was then removed and the polymer coating dried overnight by theevaporation of the dichloromethane solvent. The coated portion of thedevice was then placed into a pH 7.4 phosphate buffered saline forrelease of ACCS proteins.

Experiment 2

An approximately 1 cm×1 cm section was excised from a Dacron® vasculargraft material. It was dipped into the ACCS-ethylene vinyl acetatecopolymer suspension described above. The graft material was removed andthe polymer coating dried overnight upon the evaporation of thedichloromethane solvent. The coated portion of the device was thenplaced into a pH 7.4 phosphate buffered saline for release of ACCSproteins.

Results of Experiment 1 and 2

The results in FIG. 1 demonstrate the sustained cumulative release ofmicrograms of ACCS from the polymer coatings from the Zimaloy®prosthetic replacement hip device and from the Dacron® vascular graftover 48 hours. The coated Zimaloy® prosthetic replacement hip devicereleased a cumulative total of approximately 1303 micrograms of ACCSproteins over the 48 hour time period tested as determined by theBradford Protein Assay (Bradford, M. M. (1976), “Rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding”, Anal. Biochem. 72: 248-254). Thecoated Dacron® vascular graft material released a cumulative total ofapproximately 704 micrograms of ACCS proteins over the 48 hour periodtested. The greater release from the Zimaloy® prosthetic replacement hipdevice is due to the greater surface area of the coating compared to theDacron® vascular graft material. Furthermore, the ACCS incorporated intothe ELVAX polymer coatings were shown to contain 23 representative ACCSanalytes. Antibody array revealed the following proteins release fromthe Dacron® and Zymaloy® ELVAX/ACCS coated devices; PDGF-BB, sTNF RI,TRAIL R3, IGFBP-6, AXL, Amphiregulin, uPAR, PDGF AA, ErbB3, LYVE-1,FLRG, Adipsin/Factor D, Angiogenin, VEGF, DIPPIV, IGFBP-2, TIMP-2,TIMP-1, GDF-15, PAI-I, Decorin, CA125 and DKK-3.

Example 5 Coating of a Titanium Implant Wire with ACCS

ACCS was diafiltered using four Amicon® Ultra-15 centrifugal filterdevices (EMD Millipore Corp., Billerica, Mass.) with 12 ml each. Bufferexchange was performed using Water for Injection (WFI) following themanufacturer's instructions. The filtered ACCS solution was frozen andlyophilized for coating.

A 5% Ethylene Vinyl Acetate (EVA) solution was prepared indichloromethane. Lyophilized ACCS powder was added to the equivalent of15% wt ACCS/wt ACCS plus polymer. This mixture was then used to coatfive 10 cm length sections of titanium 6AI-4V (grade 5) implant wires.Five pre-chilled pieces of the titanium wire implants were dippedseparately into the solution and allowed to dry overnight at roomtemperature. The pieces were weighed before and after coating and showeda total weight increase of approximately 1 mg. The coated wire implantstogether were placed in a test tube containing 150 μl of WFI and allowedto elute for 24 hrs at room temperature. Several collections of releasemedia were performed after incubation times of 2 hrs, 6 hrs and 24 hrs.

Preliminary results demonstrated that 1329 μg/mL of DPPIV activity, acomponent of ACCS, was detected at 2 hrs. Not surprisingly, the 6 hr and24 hr time points were below the level of detection with this particularassay of DPPIV activity. However, the presence of luminescence wasdetected at 2 hrs and 6 hrs indicating sustained release of ACCS fromthe EVAc coating (see Table 1 below).

TABLE 1 Time (hours) Average Luminescence 0 0 2 7721 6 429

Additional, more sensitive assays such as antibody array for other ACCSproteins are currently being investigated.

Example 6 Retention of ACCS Bioactivity after Combination orIncorporation into Gelatin Film

A 5% gelatin in ACCS was prepared by dissolving gelatin in an aqueoussolution of ACCS at 40° C. for 15 min. Five percent gelatin in 1×PBS wasalso prepared as a control. Two hundred μl of 5% ACCS/gelatin was placedin a 1.5 mL centrifuge tube and allowed to air dry for 48 hrs at roomtemperature to form a transparent solid film. Control samples were alsoheld at room temperature but were not allowed to dry. On the day of theassay, the dried ACCS/gelatin film was solubilized for 4 hrs with 180 μlof water for injection (WFI). Samples were then heated to 37° C. tosolubilize the gels and then diluted into assay buffer and tested forDPPIV activity.

Table 2 below shows the results of the experiment comparing the DPPIVactivity of ACCS/gelatin, dried and solubilized ACCS/gelatin film, a 95%solution of ACCS, and 5% Phosphate Buffered Saline (PBS)/gelatinnegative control. The results showed that ACCS/gelatin retained itsactivity and that drying the solution did not reduce DPPIV activity.

TABLE 2 Formulation DPPIV activity (pg/mL) ACCS/gelatin 16,201 Dried andsolubilized ACCS/ 23,538 gelatin 95% ACCS solution 18,344 5% PBS/gelatin116

Example 7 Cumulative Release of 4 Representative ACCS Proteins fromTitanium EVAc Coated Wires Measured by MSD Antibody Array Assay

An experiment was performed to measure the cumulative release of 4representative ACCS proteins from Titanium EVAc coated wires measured byMSD antibody array assay. As shown in Table 3 below, the cumulativerelease of four representative proteins from EVAc coated titanium wireswas shown to increase over 24 hours. This indicates sustained release ofthese ACCS proteins from EVAc coated titanium wires.

TABLE 3 Protein TIMP-1 GDF-15 PAI-1 DKK3 Time (hrs) (pg/mL) (pg/mL)(pg/mL) (pg/mL) 2 248.52 60.01 1187.72 9221.26 6 277.68 62.49 1301.689501.24 24 298.89 62.49 1303.43 9503.03

Example 8 Cumulative Release of 4 Representative ACCS Proteins fromTitanium Gelatin Coated Wires Measured by MSD Antibody Array Assay

An experiment was performed to measure the cumulative release of 4representative ACCS proteins from Titanium Gelatin coated wires measuredby MSD antibody array assay. As shown in Table 4 below, the cumulativerelease of four representative proteins from Gelatin coated titaniumwires was shown to increase over 7 hours. This indicates sustainedrelease of these ACCS proteins from Gelatin coated titanium wires.

TABLE 4 Protein TIMP-1 GDF-15 PAI-1 DKK3 Time (hrs) (pg/mL) (pg/mL)(pg/mL) (pg/mL) 2 1458.85 94.35 1332.60 48.18 4 1645.15 118.81 2516.2075.65 7 1756.04 124.40 2905.85 120.19

Example 9 Inflammatory Model—Use of ACCS to Prevent Onset of PeriodontalDisease in an Animal Model

Objective:

The aim of this study was to evaluate the preventive role of ACCS inPorphyromonas gingivalis (P. gingivalis)-induced experimentalperiodontitis in rabbits

Methods:

Eight New-Zealand White rabbits were distributed into 3 groups: 1.Untreated (n=2), 2. Control (unconditioned ACCS culture media) (n=3),and 3. ACCS (n=3). At baseline, all rabbits received silk ligaturesbilaterally tied around mandibular second premolars under generalanesthesia. The assigned test materials, ACCS or control, in volumes of10 μL were topically applied to the ligated sites with a bluntneedled-Hamilton Syringe from the time of ligature; control animalsreceived ligature, but no treatment. Topical P. gingivalis-containingslurry (1 mL) was subsequently applied to induce the periodontalinflammation. The application of test materials and P. gingivaliscontinued for 6 weeks on an every-other-day schedule. At 6 weeks,following euthanasia, the mandibles were surgically harvested.Morphometric, radiographic and histologic evaluations were performed.

Results:

Macroscopic evaluations including soft tissue assessments, crestal boneand infrabony measurements showed significant periodontal breakdowninduced by P. gingivalis in control and no treatment groups at 6 weekscompared to historical ligature-alone groups (p=0.05, p=0.03,respectively). ACCS application significantly inhibited soft tissueinflammation and prevented both crestal bone loss and infrabony defectformation compared to untreated and control groups (p=0.01, p=0.05,respectively). Histologic assessments and histomorphometric measurementssupported the clinical findings; ACCS treated animals demonstratedsignificantly less inflammation in soft tissue and less bone losscompared to the untreated and control groups (p=0.05).

Conclusions:

Topical ACCS application prevents periodontal inflammatory changes andbone loss induced by P. gingivalis as shown both at clinical andhistopathological level. ACCS has potential as a therapeutic approachfor the prevention of periodontal diseases

Example 10 Inflammatory Model—Use of ACCS to Stop Progression of orReverse Periodontal Disease in an Animal Model

Objective:

The aim of this study was to evaluate the therapeutic actions of ACCS inthe treatment of periodontitis induced by P. gingivalis.

Methods:

The study was conducted using a two-phase rabbit periodontitis protocol:1—Disease induction (6 weeks) and 2—Treatment (6 weeks). Periodontaldisease was induced in 16 New-Zealand White rabbits by every-other-dayapplication of topical P. gingivalis to ligatured mandibular premolars.At the end of Phase 1, 4 randomly selected rabbits were sacrificed toserve as the baseline disease group. For Phase 2, the remaining 12rabbits were distributed into 3 groups (n=4), 1—Untreated, 2—Control(unconditioned ACCS culture media) and 3—ACCS treatment. At the end ofPhase 2, morphometric, radiographic and histologic evaluations wereperformed on harvested mandibles.

Results:

The baseline disease group exhibited experimental periodontitisevidenced by tissue inflammation and bone loss. At the end of Phase 2,the untreated group showed significant disease progression characterizedby increased soft and hard tissue destruction (p=0.05). The tissueinflammation and bone loss was significantly reduced by topical ACCScompared to baseline disease and untreated groups (p=0.05; p=0.002,respectively). The control treatment also arrested disease progressioncompared to untreated group (p=0.01), but there was no improvement inperiodontal health compared to baseline disease (p=0.4).Histopathological assessments revealed similar findings; ACCS stoppedthe progression of inflammatory process (p=0.003) and reversed bonedestruction induced by P. gingivalis (p=0.008). The ACCS-treated grouphad minimal osteoclastic activity limited to crestal area compared tountreated and control groups, which showed a profound osteoclastogenicactivity at the bone crest as well as at interproximal sites.

Conclusions:

Topical application of ACCS stopped the progression of periodontalinflammation and resulted in tissue regeneration in rabbit periodontitisindicating its potential therapeutic efficacy.

Example 11 Inflammatory Model—Evaluate the Efficacy of Topically AppliedACCS to Inhibit Irritant 12-O-Tetradecanoylphorbol-3-Acetate (TPA) SkinInflammation in Mice

Method:

Topical treatment was given twice daily to the following groups: 1.TPA+topical control; 2. TPA+ACCS; 3. TPA+clobetasol 0.05 topicalsolution (the strongest available topical corticosteroid); 4. ACCSalone; 5. No treatment (the other untreated ear was measured). Theendpoints for the study were ear thickness and ear weight at the end ofthe experiment. The thicker the ear and the more it weighs correlateswith the degree of inflammation.

Results:

Topically applied ACCS was effective at reducing the inflammationinduced by TPA. The anti-inflammatory activity of topical ACCS reachedthe same level as clobetasol (a class 1 potent topical corticosteroid)by 3 days after beginning application.

Conclusion:

ACCS has a strong anti-inflammatory effect when applied to skin.

Example 12 Inflammatory Model—Evaluate the Efficacy of IntralesionalInjection of ACCS to Inhibit Irritant (TPA) Skin Inflammation in Mice

Method:

Intralesional injection into the ear was given once daily to thefollowing groups: 1. TPA+intralesional control; 2. TPA+intralesionalACCS; 3. TPA+intralesional kenalog (10 mg/ml) (a potent intralesionalcorticosteroid); 4. ACCS intralesional injection alone; 5. Saline shaminjections to the normal untreated ear. The endpoints for the study wereear thickness and ear weight at the end of the experiment. The thickerthe ear and the more it weighs correlates with the degree ofinflammation.

Results:

Intralesional injection of ACCS was effective at reducing theinflammation induced by TPA at all time points beginning on day 2 ofdaily injections. Intralesional kenalog (10 mg/ml) injections induced ahematoma at the site of injection, which led to some inflammation andthat is why there is not a substantial difference in ear thickness whencomparing TPA+kenalog with TPA+control.

Conclusions:

Intralesional ACCS did reduce skin inflammation but the topicallyapplied ACCS in Example 1 above had a more potent effect. There was nodifference in ear weight using either ACCS or intralesional kenalogcompared with TPA+control.

Example 13 Wound Healing Model—Effects of ACCS in an Animal Model ofChronic Wound Healing

An art-accepted animal model for chronic granulating wound was used tostudy the effects of ACCS on chronic wound healing (Hayward P G, RobsonM C: Animal models of wound contraction. In Barbul A, et al: Clinicaland Experimental Approaches to Dermal and Epidermal Repair: Normal andChronic Wounds. John Wiley & Sons, New York, 1990.).

Results:

ACCS was effective in not allowing proliferation of tissue bacterialbioburden. ACCS allowed accelerated healing of the granulating woundsignificantly faster than the non-treated infected control groups.

Example 14 Arthroplasty Animal Model

An art accepted animal model for arthroplasty (see, for example,Bernthal, N. M., et al., PloS ONE, September 2010, Vol. 5, Issue 9,e12582) is used to evaluate the ACCS coated improved medical device ofthe invention and its ability to prevent and/or down-regulate theinflammatory response associated with implanted, inserted ortranscutaneously placed medical devices. This model is also used toevaluate the ACCS coated improved medical device's ability to deliver,in a controlled manner, physiologically relevant inflammatoryresponse-modulating cytokines and growth factors that are capable ofpreventing and/or down-regulating the inflammatory response associatedwith implanted, inserted or transcutaneously placed medical devices.This model is also used to evaluate the ACCS coated improved medicaldevice's ability to deliver, in a controlled manner, physiologicallyrelevant wound healing growth factors and cytokines that are capable ofpromoting healing of tissue at the medical device implantation,insertion or transcutaneous placement site in a patient.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1.-20. (canceled)
 21. An improved implantable, insertable ortranscutaneous medical device useful for delivering inflammatoryresponse-modulating cytokines and growth factors into a subject's body,wherein the improvement to the implantable, insertable or transcutaneousmedical device comprises a medical device having a coating on itssurface, wherein the coating comprises Amnion-derived Cellular CytokineSolution (ACCS) dispersed in a polymeric coating material, wherein theACCS comprises about 5.0-16 ng/mL VEGF, about 3.5-4.5 ng/mL Angiogenin,about 100-165 μg/mL PDGF, about 2.5-2.7 ng/mL TGFβ2, about 0.68 μg/mLTIMP-1 and about 1.04 μg/mL TIMP-2.
 22. The improved implantable medicaldevice of claim 21 wherein the improved implantable medical device isselected from the group consisting of vascular grafts, breast implants,and implantable cardioverter defibrillators.
 23. The improved insertablemedical device of claim 21 wherein the improved insertable medicaldevice is selected from the group consisting of tympanostomy tubes andintrauterine devices.
 24. The improved implantable, insertable ortranscutaneous medical device of claim 21 wherein the ACCS islyophilized prior to dispersal in the polymeric coating material.
 25. Animproved implantable, insertable or transcutaneous medical device usefulfor increasing the device's effectiveness and biocompatibility andreducing the device's failure rate following implantation, insertion ortranscutaneous placement into subject's body, wherein the improvement tothe medical device comprises a medical device having a coating on itssurface, wherein the coating comprises Amnion-derived Cellular CytokineSolution (ACCS) dispersed in a polymeric coating material, wherein theACCS comprises about 5.0-16 ng/mL VEGF, about 3.5-4.5 ng/mL Angiogenin,about 100-165 μg/mL PDGF, about 2.5-2.7 ng/mL TGFβ2, about 0.68 μg/mLTIMP-1 and about 1.04 μg/mL TIMP-2.
 26. The improved implantable medicaldevice of claim 25 wherein the improved implantable medical device isselected from the group consisting of vascular grafts, breast implants,and implantable cardioverter defibrillators.
 27. The improved insertablemedical device of claim 25 wherein the improved insertable medicaldevice is selected from the group consisting of tympanostomy tubes andintrauterine devices.
 28. The improved implantable, insertable ortranscutaneous medical device of claim 25 wherein the ACCS islyophilized prior to dispersal in the polymeric coating material.
 29. Animproved implantable, insertable or transcutaneous medical device usefulfor implantation, insertion or transcutaneous placement into a subject'sbody, wherein the improvement to the medical device comprises a medicaldevice having a coating on its surface, wherein the improvedimplantable, insertable or transcutaneous medical device is made by themethod comprising the steps of a) obtaining conditioned medium, whereinthe conditioned medium is made by a method comprising the steps of i)obtaining a placenta and isolating an amnion from the placenta, ii)enzymatically releasing amnion-derived epithelial cells from the amnion,iii) collecting the released amnion-derived epithelial cells, iv)culturing the collected amnion-derived epithelial cells of step (c) inbasal culture medium that is supplemented with human serum albumin andrecombinant human EGF; v) removing the culture medium after about 2-3days and applying fresh culture medium; vi) collecting the culturemedium after culturing the cells for 2-3 days; and b) dispersing theconditioned medium obtained in step (a) in a polymeric coating material;and c) coating the implantable, insertable or transcutaneous medicaldevice with the conditioned medium dispersed in the polymeric coatingmaterial of step (b).
 30. The improved implantable, insertable ortranscutaneous medical device of claim 29 wherein steps (v) and (vi) arerepeated a plurality of times.
 31. The composition of claim 29 whereinthe basal medium is IMDM.
 32. The composition of claim 31 wherein theIMDM is supplemented with 0.5% human serum albumin.
 33. The compositionof claim 29 wherein the recombinant human EGF is at a concentration of10 ng/mL of culture medium.